Eukaryotic chromatin structure and gene expression are regulated by histone acetylation by histone acetyltransferase (HAT), and deacetylation by histone deacetylase (HDAC). HDAC inhibitors are already known to induce cancer cell differentiation and apoptosis, and are expected to be useful as antitumor agents (Marks, P. A., Richon, V. M., and Rifkind, R. A. (2000). Histone deacetylase inhibitors: Inducers of differentiation or apoptosis of transformed cells. J. Natl. Cancer Inst. 92, 1210-1216; Yoshida, M., Horinouchi, S., and Beppu, T. (1995). Trichostatin A and trapoxin: novel chemical probes for the role of histone acetylation in chromatin structure and function. Bioessays 17, 423-430; Bernhard, D., Löffler, M., Hartmann, B. L., Yoshida, M., Kofler, R., and Csordas, A. (1999). Interaction between dexamethasone and butyrate in apoptosis induction: non-additive in thymocytes and synergistic in a T cell-derived leukemia cell line. Cell Death Diff. 6, 609-607).
In fact, clinical studies have begun in the United States for some HDAC inhibitors (Nakajima, H., Kim, Y. B., Terano, H., Yoshida, M., and Horinouchi, S. (1998). FR901228, a potent antitumor antibiotic, is a novel histone deacetylase inhibitor. Exp. Cell Res. 241, 126-133; Saito, A., Yamashita, T., Mariko, Y., Nosaka, Y., Tsuchiya, K., Ando, T., Suzuki, T., Tsuruo, T., and Nakanishi, O. (1999). A synthetic inhibitor of histone deacetylase, MS-27-275, with marked in vivo antitumor activity against human tumors. Proc. Natl. Acad. Sci. USA 96, 4592-4597) that are effective as antitumor agents in animal experiments.
Tricostatin A (TSA) is well known as a specific HDAC inhibitor (Yoshida, M., Kijima, M., Akita, M., and Beppu, T. (1990). Potent and specific inhibition of mammalian histone deacetylase both in vivo and in vitro by trichostatin A. J. Biol. Chem. 265, 17174-17179). Actually, TSA has been known to induce differentiation to leukemia cells, neuronal cells, breast cancer cells, and the like (Yoshida, M., Nomura, S., and Beppu, T. Effects of trichostatins on differentiation of murine erythroleukemia cells. Cancer Res. 47: 3688-3691, 1987; Hoshikawa, Y., Kijima, M., Yoshida, M., and Beppu, T. Expression of differentiation-related markers in teratocarcinoma cells via histone hyperacetylation by trichostatin A. Agric. Biol. Chem. 55: 1491-1495, 1991; Minucci, S., Horn, V., Bhattacharyya, N., Russanova, V., Ogryzko, V. V., Gabriele, L., Howard, B. H., and Ozato, K. A histone deacetylase inhibitor potentiates retinoid receptor action in embryonal carcinoma cells. Proc. Natl. Acad. Sci. USA 94: 11295-11300, 1997; Inokoshi, J., Katagiri, M., Arima, S., Tanaka, H., Hayashi, M., Kim, Y. B., Furumai, R., Yoshida, M., Horinouchi, S., and Omura, S. (1999). Neuronal differentiation of Neuro 2a cells by inhibitors of cell progression, trichostatin A and butyrolactone I. Biochem. Biophys. Res. Commun. 256, 372-376; Wang, J., Saunthararajah, Y., Redner, R. L., and Liu, J. M. Inhibitors of histone deacetylase relieve ETO-mediated repression and induce differentiation of AML1-ETO leukemia cells. Cancer Res. 59: 2766-2769, 1999; Munster, P. N., Troso-Sandoval, T., Rosen, N., Rifkind, R., Marks, P. A., and Richon, V. M. The histone deacetylase inhibitor suberoylanilide hydroxamic acid induces differentiation of human breast cancer cells. Cancer Res. 61: 8492-8497, 2001; Ferrara, F. F., Fazi, F., Bianchini, A., Padula, F., Gelmetti, V., Minucci, S., Mancini, M., Pelicci, P. G., Lo Coco, F., and Nervi, C. Histone deacetylase-targeted treatment restores retinoic acid signaling and differentiation in acute myeloid leukemia. Cancer Res. 61: 2-7, 2001; Gottlicher, M., Minucci, S., Zhu, P., Kramer, O. H., Schimpf, A., Giavara, S., Sleeman, J. P., Lo Coco, F., Nervi, C., Pelicci, P. G., and Heinzel, T. Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. EMBO J. 20: 6969-6978, 2001). Furthermore, the TSA activities of differentiation induction and apoptosis induction are known to synergistically increase when used in combination with drugs which activate gene expression by mechanisms different to HDAC inhibitors. For example, cancer cell differentiation is promoted by using HDAC inhibitors in combination with retinoic acids, which activate retinoic acid receptors that serve as nuclear receptors, inducing gene expression relevant to differentiation (Minucci, S., Horn, V., Bhattacharyya, N., Russanova, V., Ogryzko, V. V., Gabriele, L., Howard, B. H., and Ozato, K. A histone deacetylase inhibitor potentiates retinoid receptor action in embryonal carcinoma cells. Proc. Natl. Acad. Sci. USA 94: 11295-11300, 1997; Ferrara, F. F., Fazi, F., Bianchini, A., Padula, F., Gelmetti, V., Minucci, S., Mancini, M., Pelicci, P. G., Lo Coco, F., and Nervi, C. Histone deacetylase-targeted treatment restores retinoic acid signaling and differentiation in acute myeloid leukemia. Cancer Res. 61: 2-7, 2001; Coffey, D. C., Kutko, M. C., Glick, R. D., Butler, L. M., Heller, G., Rifkind, R. A., Marks, P. A., Richon, V. M., and La Quaglia, M. P. The histone deacetylase inhibitor, CBHA, inhibits growth of human neuroblastoma xenografts in vivo, alone and synergistically with all-trans retinoic acid. Cancer Res. 61: 3591-3594, 2001; Petti, M. C., Fazi, F., Gentile, M., Diverio, D., De Fabritiis, P., De Propris, M. S., Fiorini, R., Spiriti, M. A., Padula, F., Pelicci, P. G., Nervi, C., and Lo Coco, F. Complete remission through blast cell differentiation in PLZF/RARalpha-positive acute promyelocytic leukemia: in vitro and in vivo studies. Blood 100: 1065-1067, 2002). 5-azadeoxycytidine inhibits DNA methylation to reduce expression of tumor suppressor genes in many cancer cells. TSA used in combination with 5-azadeoxycytidine promotes cancer cell apoptosis and restoration of tumor-suppressing gene expression (Nan, X., Ng, H. H., Johnson, C. A., Laherty, C. D., Turner, B. M., Eisenman, R. N., and Bird, A. Transcriptional repression by deacetylase complex. Nature 393: 386-389, 1998; Cameron, E. E., Bachman, K. E., Myohanen, S., Herman, J. G., and Baylin, S. B. Synergy of demethylation and histone deacetylase inhibition in the re-expression of genes silenced in cancer. Nature Genet. 21: 103-107, 1999; Li, Q. L., Ito, K., Sakakura, C., Fukamachi, H., Inoue, K., Chi, X. Z., Lee, K. Y., Nomura, S., Lee, C. W., Han, S. B., Kim, H. M., Kim, W. J., Yamamoto, H., Yamashita, N., Yano, T., Ikeda, T., Itohara, S., Inazawa, J., Abe, T., Hagiwara, A., Yamagishi, H., Ooe, A., Kaneda, A., Sugimura, T., Ushijima, T., Bae, S. C., and Ito, Y. Causal relationship between the loss of RUNX3 expression and gastric cancer. Cell 109: 113-124, 2002; Boivin, A. J., Momparler, L. F., Hurtubise, A., and Momparler, R. L. Antineoplastic action of 5-aza-2′-deoxycytidine and phenylbutyrate on human lung carcinoma cells. Anticancer Drugs 13: 869-874, 2002; Primeau, M., Gagnon, J., and Momparler, R. L. Synergistic antineoplastic action of DNA methylation inhibitor 5-AZA-2′-deoxycytidine and histone deacetylase inhibitor depsipeptide on human breast carcinoma cells. Int J Cancer 103: 177-184, 2003).
HDAC inhibitors are expected to be not only antitumor agents but also cancer preventives. TSA, SAHA, and the like significantly suppressed the occurrence of breast cancer induced in animal models. Also, investigations carried out using valproic acids indicated that HDAC inhibitors suppress metastasis (Gottlicher, M., Minucci, S., Zhu, P., Kramer, O. H., Schimpf, A., Giavara, S., Sleeman, J. P., Lo Coco, F., Nervi, C., Pelicci, P. G., and Heinzel, T. Valproic acid defines a novel class of HDAC inhibitors inducing differentiation of transformed cells. EMBO J. 20: 6969-6978, 2001).
HDAC inhibitors are used not only as tumor suppressive agents, but also, for example, as agents for treating and improving autoimmune diseases, skin diseases, infectious diseases, and such (Darkin-Rattray et al. Proc. Natl. Acad. Sci. USA 93, 13143-13147, 1996), as well as in improving the efficiency of vector introduction in gene therapy (Dion et al., Virology 231, 201-209, 1997), promoting the expression of introduced genes (Chen et al., Proc. Natl. Acad. Sci. USA 94, 5798-5803, 1997), and the like. Furthermore, HDAC inhibitors are presumed to have angiogenesis-inhibiting functions (Kim, M. S., Kwon, H. J., Lee, Y. M., Baek, J. H., Jang, J. E., Lee, S. W., Moon, E. J., Kim, H. S., Lee, S. K., Chung, H. Y., Kim, C. W., and Kim, K. W. (2001). Histone deacetylases induce angiogenesis by negative regulation of tumor suppressor genes. Nature Med. 7, 437-443; Kwon, H. J., Kim, M. S., Kim, M. J., Nakajima, H., and Kim, K. W. (2002). Histone deacetylase inhibitor FK228 inhibits tumor angiogenesis. Int. J. Cancer 97, 290-296).
Ten or more HDAC subtypes exist, and recently, specific HDAC subtypes have been identified as being closely related to cancers. For example, it has been discovered that acetylation of the tumor suppressor gene p53, which plays an extremely important role in suppressing carcinogenesis, is very important in the functional expression of p53 itself (Ito, A., Lai, C. H., Zhao, X., Saito, S., Hamilton, M. H., Appella, E., and Yao, T. P. (2001). p300/CBP-mediated p53 acetylation is commonly induced by p53-activating agents and inhibited by MDM2. EMBO J. 20, 1331-1340), and HDAC1 and HDAC2 are involved in the inhibition of p53 function (Juan, L. J., Shia, W. J., Chen, M. H., Yang, W. M., Seto, E., Lin, Y. S., and Wu, C. W. (2000). Histone Deacetylases Specifically Down-regulate p53-dependent Gene Activation. J. Biol. Chem. 275, 20436-20443). It has also been discovered that proteins PML-RAR and PLZF-RAR, involved in the onset of promyelocytic leukemia (APL), and oncogenes such as Bcl-6, which is involved in the onset of lymphomas, recruit HDAC4 or such via nuclear co-repressors, and suppress expression of the gene group necessary for normal differentiation, causing carcinogenesis (Dhordain P., Albagli, O., Lin, R. J., Ansieau, S., Quief, S., Leutz, A., Kerckaert, J. P., Evans, R. M., and Leprince, D. (1997). Corepressor SMRT binds the BTB/POZ repressing domain of the LAZ3/BCL6 oncoprotein. Proc. Natl. Acad. Sci. USA 94, 10762-10767; Grignani, F., De, M. S., Nervi, C., Tomassoni, L., Gelmetti, V., Cioce, M., Fanelli, M., Ruthardt, M., Ferrara, F. F., Zamir, I., Seiser, C., Grignani, F., Lazar, M. A., Minucci, S., and Pelicci, P. G. (1998). Fusion proteins of the retinoic acid receptor-alpha recruit histone deacetylase in promyelocytic leukaemia. Nature 391, 815-818; He, L. Z., Guidez, F., Tribioli, C., Peruzzi, D., Ruthardt, M., Zelent, A., and Pandolfi, P. P. (1998). Distinct interactions of PML-RARalpha and PLZF-RARalpha with co-repressors determine differential responses to RA in APL. Nature Genet. 18, 126-135; Lin, R. J., Nagy, L., Inoue, S., Shao, W., Miller, W. J., and Evans, R. M. (1998). Role of the histone deacetylase complex in acute promyelocytic leukaemia. Nature 391, 811-814). On the other hand, HDAC subtypes which play a very important role in the development and differentiation of normal tissues are known to exist among those HDAC subtypes with tissue-specific expression (McKinsey, T. A., Zhang, C. L., Lu, J., and Olson, E. N. (2000). Signal-dependent nuclear export of a histone deacetylase regulates muscle differentiation. Nature 408, 106-111; Verdel, A., and Khochbin, S. (1999). Identification of a new family of higher eukaryotic histone deacetylases. Coordinate expression of differentiation-dependent chromatin modifiers. J. Biol. Chem. 274, 2440-2445). In order to avoid inhibition of these HDACs, development of a subtype-specific inhibitor is thought to be necessary.
HDAC6 is an enzyme which is shuttled between the nucleus and the cytoplasm by nucleo-cytoplasmic transport, and which normally locates in the cytoplasm (Verdel, A., Curtet, S., Brocard, M. -P., Rousseaux, S., Lemercier, C., Yoshida, M., and Khochbin, S. (2000). Active maintenance of mHDA2/mHDAC6 histone-deacetylase in the cytoplasm. Curr. Biol. 10, 747-749). HDAC6 is highly expressed in the testes, and is presumed to relate to the differentiation of normal tissues. Furthermore, HDAC6 is known to be associated with microtubule deacetylation, and to control microtubule stability (Matsuyama, A., Shimazu, T., Sumida, Y., Saito, A., Yoshimatsu, Y., Seigneurin-Berny, D., Osada, H., Komatsu, Y., Nishino, N., Khochbin, S., Horinouchi, S., and Yoshida, M. (2002). In vivo destabilization of dynamic microtubules by HDAC6-mediated deacetylation. EMBO J. 21, 6820-6831). HDAC6 is also a deacetylation enzyme bonded to a microtubule and affecting cell mobility (Hubbert, C., Guardiola, A., Shao, R., Kawaguchi, Y., Ito, A., Nixon, A., Yoshida, M., Wang, X. -F., and Yao, T. -P. (2002). HDAC6 is a microtubule-associated deacetylase. Nature 417, 455-458). Accordingly, HDAC6 inhibitors may be metastasis-suppressing agents. TSA inhibits each HDAC subtype to about the same degree. However, HDAC6 cannot be inhibited by trapoxins comprising cyclic tetrapeptide structure and epoxyketone as active groups (Furumai, R., Komatsu, Y., Nishino, N., Khochbin, S., Yoshida, M., and Horinouchi, S. Potent histone deacetylase inhibitors built from trichostatin A and cyclic tetrapeptide antibiotics including trapoxin. Proc. Natl. Acad. Sci. USA 98: 87-92, 2001). Based on the information on the three-dimensional structure of the enzyme, trapoxins are assumed to exert poor binding properties to HDAC6 due to the structure of its cyclic tetrapeptide moiety that interacts with the weakly conserved outward surface of the enzyme active center. This implies that altering the cyclic tetrapeptide portion may result in inhibitors that are selective for a variety of HDAC.
TSA shows inhibition activity due to the coordination of its hydroxamic acid group with zinc in the HDAC active pocket (Finnin, M. S., Donigian, J. R., Cohen, A., Richon, V. M., Rifkind, R. A., Marks, P. A., Breslow, R., and Pavletich, N. P. Structures of a histone deacetylase homologue bound to the TSA and SAHA inhibitors. Nature 401: 188-193, 1999). Examples of known HDAC inhibitors comprising hydroxamic acid are Oxamflatin (Kim, Y. B., Lee, K. -H., Sugita, K., Yoshida, M., and Horinouchi, S. Oxamflatin is a novel antitumor compound that inhibits mammalian histone deacetylase. Oncogene 18: 2461-2470, 1999) and CHAP (Furumai, R., Komatsu, Y., Nishino, N., Khochbin, S., Yoshida, M., and Horinouchi, S. Potent histone deacetylase inhibitors built from trichostatin A and cyclic tetrapeptide antibiotics including trapoxin. Proc. Natl. Acad. Sci. USA 98: 87-92, 2001., Komatsu, Y., Tomizaki, K. -y., Tsukamoto, M., Kato, T., Nishino, N., Sato, S., Yamori, T., Tsuruo, T., Furumai, R., Yoshida, M., Horinouchi, S., and Hayashi, H. Cyclic Hydroxamic-acid-containing Peptide 31, a potent synthetic histone deacetylase inhibitor with antitumor activity. Cancer Res. 61: 4459-4466, 2001). However, since TSA is instable in blood and has a strong hydroxamic acid chelating function, it chelates with other essential metal ions, and therefore, HDAC inhibitors comprising hydroxamic acid have not actually been used as antitumor agents to date. Meanwhile, thiol groups produced by the reduction of FK228 disulfide bonds have recently been shown to serve as active groups to be coordinated with zinc in the HDAC active pocket, inhibiting HDAC. Thus, FK228 is a prodrug that is activated when reduced by cellular reducing activity (Furumai, R., Matsuyama, A., Kobashi, N., Lee, K. -H., Nishiyama, M., Nakajima, H., Tanaka, A., Komatsu, Y., Nishino, N., Yoshida, M., and Horinouchi, S. (2002). FK228 (depsipeptide) as a natural prodrug that inhibits class I histone deacetylases. Cancer Res. 62, 4916-4921).
Furthermore, a number of HDAC inhibitors comprising cyclic tetrapeptide structures and epoxyketones as active groups have been isolated from natural environments. On the basis of such findings, the cyclic tetrapeptide structure is suggested to be useful in enzyme identification (as described above, Yoshida, et al., 1995), however, from various viewpoints such as stability, existing inhibitors have not advanced to the level of being satisfactorily qualified as pharmaceutical products. Therefore, production of pharmaceutical agents in which these problematic points have been resolved is strongly anticipated.